Imaging Agents

ABSTRACT

The present invention provides novel amino acid compounds useful in detecting and evaluating brain and body tumors. These compounds have the advantageous properties of rapid uptake and prolonged retention in tumors and can be labeled with halogen isotopes such as fluorine-18, iodine-123, iodine-124, iodine-125, iodine-131, bromine-75, bromine-76, bromine-77, bromine-82, astatine-210, astatine-211, and other astatine isotopes. These compounds can also be labeled with technetium and rhenium isotopes using known chelation complexes. The compounds disclosed herein bind tumor tissues in vivo with high specificity and selectivity when administered to a subject. Preferred compounds show a target to non-target ratio of at least 2:1, are stable in vivo and substantially localized to target within 1 hour after administration. Preferred compounds include 1-amino-2-[ 18 F]fluorocyclobutyl-1-carboxylic acid (2-[ 18 F]FACBC) and 1-amino-2-[ 18 F]fluoromethylcyclobutyl-1-carboxylic acid (2-[ 18 F]FMACBC). The labeled amino acid compounds of the invention are useful as imaging agents in detecting and/or monitoring tumors in a subject by PET or SPECT.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Applications No.60/693,385, filed Jun. 23, 2005 and No. 60/728,082, filed Oct. 19, 2005,which are incorporated herein in their entirety to the extent notinconsistent herewith.

ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT

This invention was made with government support under Grant No.5-R21-CA-098891 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

This invention relates to novel amino acid analogs having specific andselective binding in a biological system, particularly brain andsystemic tumors, and capable of being used for positron emissiontomography (PET) and single photon emission (SPECT) imaging methods.

The development of radiolabeled amino acids for use as metabolic tracersto image tumors using positron emission tomography (PET) and singlephoton emission computed tomography (SPECT) has been underway for sometime. Although radiolabeled amino acids have been applied to a varietyof tumor types, their application to intracranial tumors has receivedconsiderable attention due to potential advantages over other imagingmodalities. After surgical resection and/or radiotherapy of braintumors, conventional imaging methods such as CT and MRI do not reliablydistinguish residual or recurring tumor from tissue injury due to theintervention and are not optimal for monitoring the effectiveness oftreatment or detecting tumor recurrence [Buonocore, E (1992), ClinicalPositron Emission Tomography. Mosby-Year Book, Inc. St. Louis, Mo., pp17-22; Langleben, D D et al. (2000), J. Nucl. Med. 41:1861-1867].

The leading PET agent for diagnosis and imaging of neoplasms,2-[¹⁸F]fluorodeoxyglucose (FDG), has limitations in the imaging of braintumors. Normal brain cortical tissue shows high [¹⁸F]FDG uptake as doesinflammatory tissue which can occur after radiation or surgical therapy;these factors can complicate the interpretation of images acquired with[¹⁸F]FDG [Griffeth, L K et al. (1993), Radiology. 186:37-44; Conti, P S(1995)].

A number of reports indicate that PET and SPECT imaging withradiolabeled amino acids better define tumor boundaries within normalbrain than CT or MRI allowing better planning of treatment [Ogawa, T etal. (1993), Radiology. 186: 45-53; Jager, P L et al. (2001), Nucl. Med.,42:432-445]. Additionally, some studies suggest that the degree of aminoacid uptake correlates with tumor grade, which could provide importantprognostic information [Jager, P L et al. (2001) J. Nucl. Med.42:432-445].

Amino acids are required nutrients for proliferating tumor cells. Avariety of amino acids containing the positron emitting isotopescarbon-11 and fluorine-18 have been prepared. They have been evaluatedfor potential use in clinical oncology for tumor imaging in patientswith brain and systemic tumors and may have superior characteristicsrelative to 2-[¹⁸F]FDG in certain cancers. These amino acid candidatescan be subdivided into two major categories. The first category isrepresented by radiolabeled naturally occurring amino acids such as[¹¹C]valine, L-[¹¹C]leucine, L-[¹¹C]methionine (MET) andL-[1-¹¹C]tyrosine, and structurally similar analogues such as2-[¹⁸F]fluoro-L-tyrosine and 4-[¹⁸F]fluoro-L-phenylalanine. The movementof these amino acids across tumor cell membranes predominantly occurs bycarrier mediated transport by the sodium-independent leucine type “L”amino acid transport system. The increased uptake and prolongedretention of these naturally occurring radiolabeled amino acids intotumors in comparison to normal tissue is due in part to significant andrapid regional incorporation into proteins. Of these radiolabeled aminoacids, [¹¹C]MET has been most extensively used clinically to detecttumors. Although [¹¹C]MET has been found useful in detecting brain andsystemic tumors, it is susceptible to in vivo metabolism throughmultiple pathways, giving rise to numerous radiolabeled metabolites.Thus, graphical analysis with the necessary accuracy for reliablemeasurement of tumor metabolic activity is not possible. Studies ofkinetic analysis of tumor uptake of [¹¹C]MET in humans strongly suggestthat amino acid transport may provide a more sensitive measurement oftumor cell proliferation than protein synthesis.

The shortcomings associated with [¹¹C]MET may be overcome with a secondcategory of amino acids. These are non-natural amino acids such as1-aminocyclobutane-1-[¹¹C]carboxylic acid ([¹¹C]ACBC). The advantage of[¹¹C]ACBC in comparison to [¹¹C]MET is that it is not metabolized. Asignificant limitation in the application of carbon-11 amino acids forclinical use is the short 20-minute half-life of carbon-11. The20-minute half-life requires an on-site particle accelerator forproduction of the carbon-11 amino acid. In addition only a single orrelatively few doses can be generated from each batch production of thecarbon-11 amino acid. Therefore carbon-11 amino acids are poorcandidates for regional distribution for widespread clinical use.

In order to overcome the physical half-life limitation of carbon-11, wehave recently focused on the development of several new fluorine-18labeled non-natural amino acids, some of which have been disclosed inU.S. Pat. Nos. 5,808,146 and 5,817,776, both of which are incorporatedherein by reference. These includeanti-1-amino-3-[¹⁸F]fluorocyclobutyl-1-carboxylic acid(anti-[¹⁸F]FACBC), syn-1-amino-3-[¹⁸F]fluorocyclobutyl-1-carboxylic acid(syn-[¹⁸F]FACBC), syn- andanti-1-amino-3-[¹⁸F]fluoromethyl-cyclobutane-1-carboxylic acid (syn- andanti-[¹⁸F]FMACBC). These fluorine-18 amino acids can be used to imagebrain and systemic tumors in vivo based upon amino acid transport withthe imaging technique Positron Emission Tomography (PET). Ourdevelopment involved fluorine-18 labeled cyclobutyl amino acids thatmove across tumor capillaries by carrier-mediated transport involvingprimarily the “L” type large, neutral amino acid and to a lesser extentthe “A” type amino acid transport systems. Our preliminary evaluation ofcyclobutyl amino acids labeled with positron emitters, which areprimarily substrates for the “L” transport system, has shown excellentpotential in clinical oncology for tumor imaging in patients with brainand systemic tumors. The primary reasons for proposing ¹⁸F-labeling ofcyclobutyl/branched amino acids instead of ¹¹C (t_(1/2)=20 min.) are thesubstantial logistical and economic benefits gained with using ¹⁸Finstead of ¹¹C-labeled radiopharmaceuticals in clinical applications.The advantage of imaging tumors with ¹⁸F-labeled radiopharmaceuticals ina busy nuclear medicine department is primarily due to the longerhalf-life of ¹⁸F (t_(1/2)=110 min.). The longer half-life of 18F allowsoff-site distribution and multiple doses from a single production lot ofradio tracer. In addition, these non-metabolized amino acids may alsohave wider application as imaging agents for certain systemic solidtumors that do not image well with 2-[¹⁸F]FDG PET. WO 03/093412, whichis incorporated herein by reference, further discloses examples offluorinated analogs of α-aminoisobutyric acid (AIB) such as2-amino-3-fluoro-2-methylpropanoic acid (FAMP) and3-fluoro-2-methyl-2-(methylamino)propanoic acid (N-MeFAMP) suitable forlabeling with ¹⁸F and use in PET imaging. AIB is a nonmetabolizableα,α-dialkyl amino acid that is actively transported into cells primarilyvia the A-type amino acid transport system. System A amino acidtransport is increased during cell growth and division and has also beenshown to be upregulated in tumor cells [Palacin, M et al. (1998),Physiol. Rev. 78: 969-1054; Bussolati, O et al. (1996), FASEB J.10:920-926]. Studies of experimentally induced tumors in animals andspontaneously occurring tumors in humans have shown increased uptake ofradiolabeled AIB in the tumors relative to normal tissue [Conti, P S etal. (1986), Eur. J. Nucl. Med. 12:353-356; Uehara, H et al. (1997), J.Cereb. Blood Flow Metab. 17:1239-1253]. The N-methyl analog of AIB,N-MeAIB, shows even more selectivity for the A-type amino acid transportsystem than AIB [Shotwell, M A et al. (1983), Biochim. Biophys. Acta.737:267-84]. N-MeAIB has been radiolabeled with carbon-11 and ismetabolically stable in humans [Någren, K et al. (2000), J. LabelledCpd. Radiopharm. 43:1013-1021].

Although some of the amino acid analogs mentioned above are currentlybeing evaluated as tumor imaging agents in patients with brain andsystemic tumors, there is a continued need for a novel imaging agentwhich can bind tumor cells or tissues with high specificity andselectivity and can readily be prepared in sufficient quantities fortumor imaging with PET and SPECT. As a candidate compound makes thetransition from validation studies in cells in vitro and animal modelsto application in humans, the synthetic methods employed must be adaptedto allow routine, reliable production of the compound in largequantities. Towards this end, the present application discloses a seriesof novel amino acid compositions, methods of synthesizing and usingthose compounds for tumor imaging with PET and SPECT.

SUMMARY OF THE INVENTION

The present invention provides novel amino acid compounds useful indetecting and evaluating brain and systemic tumors and other uses. Thesecompounds combine the advantageous properties of1-amino-cycloalkyl-1-carboxylic acids, namely, their rapid uptake andprolonged retention in tumors with the properties of halogensubstituents, including certain useful halogen isotopes such asfluorine-18, iodine-123, isodine-124, iodine-125, iodine-131,bromine-75, bromine-76, bromine-77, bromine-82, astatine-210,astatine-211, and other astatine isotopes. In addition, the compoundscan be labeled with technetium and rhenium isotopes using knownchelation complexes.

The amino acid compounds of the invention have the following generalformula:

The invention also includes compounds represented by the followingformula:

The amino acid compounds of the invention bind target tumor tissues orcells with high specificity and selectivity when administered to asubject in vivo. Preferred amino acid compounds show a target tonon-target ratio of at least 2:1, are stable in vivo and substantiallylocalized to target within 1 hour after administration. Because of theirhigh specificity and selectivity for tumor tissues, the inventivecompounds can also be used in delivering a therapeutic agent to a giventumor site. Preferred amino acid compounds include (1S*,2R*)- and(1R*,2S*)-1-amino-2-[¹⁸F]fluorocyclobutyl-1-carboxylic acid(2-[¹⁸F]FACBC), (1S*,2R*)- and (1R*,2S*)- and (1S*,2S*)- and(1R*,2R*)1-amino-2-[¹⁸F]fluoromethylcyclobutyl-1-carboxylic acid(2-FMACBC).

Any of F, Cl, Br, I or C in the formulas I and II shown above may be instable isotopic or radioisotopic form. Particularly useful radioisotopiclabels are ¹⁸F, ¹²³I, ¹²⁵I, ¹³¹I, ⁷⁶Br, ⁷⁷Br and ¹¹C. The compounds ofthe invention can also be labeled with technetium and rhenium.Technetium-99m is known to be a useful radionuclide for SPECT imaging.The cyclic amino acids of the invention are joined to a Tc-99m metalcluster through a 4-6 carbon chain which can be saturated or possess adouble or triple bond. The Tc-99m metal cluster can be, for example, analkylthiolato complex, a cytectrene or a hydrazino nicotinamide complex(HYNIC). U.S. Pat. No. 5,817,776 describes various methods ofsynthesizing [Tc-99 m] technetium containing compounds in detail, whichis incorporated herein in its entirety.

The inventive compounds labeled with an appropriate radioisotope areuseful for tumor imaging with PET and/or SPECT, which can serve asdiagnostic purposes or evaluating efficacy of any therapeutic compoundsfor a given tumor. The inventive method of imaging a tumor comprises (a)introducing into a subject a detectable quantity of a labeled compoundof formula 1 or II or a pharmaceutically acceptable salt, ester or amidethereof; (b) allowing sufficient time for the labeled compound to becomeassociated with tumor tissue; and (c) detecting the labeled compoundassociated with the tumor with PET or SPECT.

The present invention also provides diagnostic compositions comprising aradiolabeled compound of formula I or II and optionally apharmaceutically acceptable carrier or diluent. Also within the scope ofthe invention are pharmaceutical compositions which comprise a compoundof formula I or II and optionally a pharmaceutically acceptable carrieror diluent. The pharmaceutical compositions are useful for delivering atherapeutic agent to a specific tumor site in a subject.

Also provided herein are methods of making the compounds of formulas Iand II. The synthetic strategy disclosed herein utilizes a cyclicsulfamidate precursor which can be converted to a final product (e.g.2-FACBC) with greater than 30% decay corrected yield (non-optimized).The high yield of this synthetic strategy is a major advantage whichenables sufficient quantities of the compounds of formula I or II,particularly 2-[¹⁸F]FACBC, available for tumor imaging in contrast tothe synthetic strategy for 3-FACBC. The cyclic sulfamidate radiolabelingprecursors are more reactive and less moisture-sensitive thantrifluoromethane sulfonic ester radiolabeling precursors which arecommonly used in the synthesis of anti- and syn-3-[¹⁸F]FACBC.Furthermore, because the 2-position of the1-amino-cyclobutyl-1-carboxylic acid is a neopentyl carbon which is notsusceptible to SN2 halogen substitution, cyclic sulfamidateradiolabeling precursors are required for F-18 fluoride labeling at the2-position. Accordingly, the cyclic sulfamidate precursors having thefollowing formula are also within the scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to novel amino acid compounds useful for tumorimaging and method of making and using such compounds.

In general the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in the context ofthe invention.

The term “pharmaceutically acceptable salt” as used herein refers tothose carboxylate salts or acid addition salts of the compounds of thepresent invention which are suitable for use in contact with the tissuesof patients without undue toxicity, irritation, allergic response, andthe like, commensurate with a reasonable benefit/risk ratio, andeffective for their intended use, as well as the zwitterionic forms,where possible, of the compounds of the invention. The term“pharmaceutically acceptable salt” as used herein in general refers tothe relatively nontoxic, inorganic and organic acid addition salts ofcompounds of the present invention. Also included are those saltsderived from non-toxic organic acids such as aliphatic mono anddicarboxylic acids, for example acetic acid, phenyl-substituted alkanoicacids, hydroxy alkanoic and alkanedioic acids, aromatic acids, andaliphatic and aromatic sulfonic acids. These salts can be prepared insitu during the final isolation and purification of the compounds or byseparately reacting the purified compound in its free base form with asuitable organic or inorganic acid and isolating the salt thus formed.Further representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate,palmitate, stearate, laurate, borate, benzoate, lactate, phosphate,tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate,mesylate, glucoheptonate, lactiobionate and laurylsulphonate salts,propionate, pivalate, cyclamate, isethionate, and the like. These mayinclude cations based on the alkali and alkaline earth metals, such assodium, lithium, potassium, calcium, magnesium, and the like, as wellas, nontoxic ammonium, quaternary ammonium and amine cations including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. See, for example, Berge S. M, et al., PharmaceuticalSalts, J. Pharm. Sci. 66:1-19 (1977) which is incorporated herein byreference.

Similarly, the term, “pharmaceutically acceptable carrier,” as usedherein, is an organic or inorganic composition which serves as acarrier/stabilizer/diluent of the active ingredient of the presentinvention in a pharmaceutical or diagnostic composition. In certaincases, the pharmaceutically acceptable carriers are salts. Furtherexamples of pharmaceutically acceptable carriers include but are notlimited to water, phosphate-buffered saline, saline, pH controllingagents (e.g. acids, bases, buffers), stabilizers such as ascorbic acid,isotonizing agents (e.g. sodium chloride), aqueous solvents, a detergent(ionic and non-ionic) such as polysorbate or TWEEN 80.

The term “alkyl” as used herein by itself or as part of another grouprefers to a saturated hydrocarbon which may be linear, branched orcyclic of up to 10 carbons, preferably 6 carbons, more preferably 4carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, andisobutyl. The alkyl groups disclosed herein also include optionallysubstituted alkyl groups where one or more C atoms in backbone arereplaced with a heteroatom, one or more H atoms are replaced withhalogen or —OH. The term “aryl” as employed herein by itself or as partof another group refers to monocyclic or bicyclic aromatic groupscontaining from 5 to 12 carbons in the ring portion, preferably 6-10carbons in the ring portion, such as phenyl, naphthyl ortetrahydronaphthyl. Aryl groups may be substituted with one or morealkyl groups which may be linear, branched or cyclic. Aryl groups mayalso be substituted at ring positions with substituents that do notsignificantly detrimentally affect the function of the compound orportion of the compound in which it is found. Substituted aryl groupsalso include those having heterocyclic aromatic rings in which one ormore heteroatoms (e.g., N, O or S, optionally with hydrogens orsubstituents for proper valence) replace one or more carbons in thering.

“Acyl” group is a group which includes a —CO— group.

The term “alkoxy” is used herein to mean a straight or branched chainalkyl radical, as defined above, unless the chain length is limitedthereto, bonded to an oxygen atom, including, but not limited to,methoxy, ethoxy, n-propoxy, isopropoxy, and the like. Preferably thealkoxy chain is 1 to 6 carbon atoms in length, more preferably 1-4carbon atoms in length.

The term “monoalkylamine” as used herein by itself or as part of anothergroup refers to an amino group which is substituted with one alkyl groupas defined above.

The term “dialkylamine” as employed herein by itself or as part ofanother group refers to an amino group which is substituted with twoalkyl groups as defined above.

The term “halo” employed herein by itself or as part of another grouprefers to chlorine, bromine, fluorine or iodine.

The term “heterocycle” or “heterocyclic ring”, as used herein exceptwhere noted, represents a stable 5- to 7-membered mono-heterocyclic ringsystem which may be saturated or unsaturated, and which consists ofcarbon atoms and from one to three heteroatoms selected from the groupconsisting of N, O, and S, and wherein the nitrogen and sulfurheteroatom may optionally be oxidized. Especially useful are ringscontain one nitrogen combined with one oxygen or sulfur, or two nitrogenheteroatoms. Examples of such heterocyclic groups include piperidinyl,pyrrolyl, pyrrolidinyl, imidazolyl, imidazlinyl, imidazolidinyl,pyridyl, pyrazinyl, pyrimidinyl, oxazolyl, oxazolidinyl, isoxazolyl,isoxazolidinyl, thiazolyl, thiazolidinyl, isothiazolyl, homopiperidinyl,homopiperazinyl, pyridazinyl, pyrazolyl, and pyrazolidinyl, mostpreferably thiamorpholinyl, piperazinyl, and morpholinyl.

The term “heteroatom” is used herein to mean an oxygen atom (“O”), asulfur atom (“S”) or a nitrogen atom (“N”). It will be recognized thatwhen the heteroatom is nitrogen, it may form an NR^(a)R^(b) moiety,wherein R^(a) and R^(b) are, independently from one another, hydrogen orC₁₋₄ alkyl, C₂₋₄ aminoalkyl, C₁₋₄ halo alkyl, halo benzyl, or R^(a) andR^(b) are taken together to form a 5- to 7-member heterocyclic ringoptionally having O, S or NR^(c) in said ring, where R^(c) is hydrogenor C₁₋₄ alkyl.

The compounds of the invention are useful as tumor binding agents and asNMDA receptor-binding ligands, and in radio-isotopic form are especiallyuseful as tracer compounds for tumor imaging techniques, including PETand SPECT imaging. Where X is At, the compounds have utility forradio-therapy. Particularly useful as an imaging agent are thosecompounds labeled with F-18 since F-18 has a half-life of 110 minutes,which allows sufficient time for incorporation into a radio-labeledtracer, for purification and for administration into a human or animalsubject. In addition, facilities more remote from a cyclotron, up toabout a 200 mile radius, can make use of F-18 labeled compounds.

SPECT imaging employs isotope tracers that emit high energy photons(γ-emitters). The range of useful isotopes is greater than for PET, butSPECT provides lower three-dimensional resolution. Nevertheless, SPECTis widely used to obtain clinically significant information about analogbinding, localization and clearance rates. A useful isotope for SPECTimaging is [¹²³I], a γ-emitter with a 13.3 hour half life. Compoundslabeled with [^(123I)] can be shipped up to about 1000 miles from themanufacturing site, or the isotope itself can be transported for on-sitesynthesis. Eighty-five percent of the isotope's emissions are 159 KeVphotons, which is readily measured by SPECT instrumentation currently inuse.

Accordingly, the compounds of the invention can be rapidly andefficiently labeled with [¹²³I] for use in SPECT analysis as analternative to PET imaging. Furthermore, because of the fact that thesame compound can be labeled with either isotope, it is possible tocompare the results obtained by PET and SPECT using the same tracer.

Other halogen isotopes can serve for PET or SPECT imaging, or forconventional tracer labeling. These include ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and ⁸²Br ashaving usable half-lives and emission characteristics. In general, thechemical means exist to substitute any halogen moiety for the describedisotopes. Therefore, the biochemical or physiological activities of anyhalogenated homolog of the compounds of the invention are now availablefor use by those skilled in the art, including stable isotope halogenhomologs. Astatine can be substituted for other halogen isotopes,[²¹⁰At] emits alpha particles with a half-life of 8.3 h. At-substitutedcompounds are therefore useful for tumor therapy, where binding issufficiently tumor-specific.

The invention provides methods for tumor imaging using PET and SPECT.The methods entail administering to a subject (which can be human oranimal, for experimental and/or diagnostic purposes) an image-generatingamount of a compound of the invention, labeled with the appropriateisotope and then measuring the distribution of the compound by PET if[¹⁸F] or other positron emitter is employed, or SPECT if [¹²³I] or othergamma emitter is employed. An image-generating amount is that amountwhich is at least able to provide an image in a PET or SPECT scanner,taking into account the scanner's detection sensitivity and noise level,the age of the isotope, the body size of the subject and route ofadministration, all such variables being exemplary of those known andaccounted for by calculations and measurements known to those skilled inthe art without resort to undue experimentation.

It will be understood that compounds of the invention can be labeledwith an isotope of any atom or combination of atoms in the structure.While [¹⁸F], and [¹²⁵I] have been emphasized herein as beingparticularly useful for PET, SPECT and tracer analysis, other uses arecontemplated including those flowing from physiological orpharmacological properties of stable isotope homologs and will beapparent to those skilled in the art.

The compounds of the invention can also be labeled with technetium (Tc)via Tc adducts. Isotopes of Tc, notably Tc^(99m), have been used fortumor imaging. The present invention provides Tc-complexed adducts ofcompounds of the invention, which are useful for tumor imaging. Theadducts are Tc-coordination complexes joined to the cyclic amino acid bya 4-6 carbon chain which can be saturated or possess a double or triplebond. Where a double bond is present, either E (trans) or Z (cis)isomers can be synthesized, and either isomer can be employed. Theinventive compounds labeled with Tc are synthesized by incorporating the^(99m)Tc isotope as a last step to maximize the useful life of theisotope.

The amino acid compounds of the invention have the following generalstructure:

The amino acid compounds of the above formula are synthesized inspecialized, non-standard routes to maximize a useful lifetime forshort-lived isotopes (i.e., last step incorporation of isotopes), and tomaximize yield and purity, as described below. Scheme 1 exemplifies thesynthesis of 2-[¹⁸F]FACBC, i.e.,(S)-(−)-anti-1-amino-2-[¹⁸F]fluorocyclobutyl-1-carboxylic acid((S)-(−)-anti-2-[¹⁸F]FACBC)) and(R)-(+)-anti-1-amino-2-[¹⁸F]fluorocyclobutyl-1-carboxylic acid((R)-(+)-anti-2-[¹⁸F]FACBC)).

The key feature of synthetic scheme 1 is the use of cyclic sulfamidateprecursors such as la and lb to enable incorporation of F-18 fluorideonto the 2-position in high radiochemical yield and subsequent efficientconversion into 2-[¹⁸F]FACBC.

The synthetic strategy outlined in scheme 1 is particularly advantageousin that approximately 2-5 mg of shelf stable cyclic sulfamidateprecursor la and lb can provide greater than 30% decay corrected yieldof the final product [¹⁸F]22 in over 99% radiochemical purity. This isin good contrast with the synthetic strategy used to synthesizeanti-3-[¹⁸F]FACBC (see scheme 2 below) [McConathy et al. (2003) Journalof Applied Radiation and Isotopes 28:657-666]. According to thesynthetic steps shown in scheme 2, approximately 20 mg of 10 must beused to obtain a reasonable conversion (˜24% radiochemical yield (RCY))of compound 10 into anti-3-[¹⁸F]FACBC 12. In contrast, the amino acidcompounds of the invention can readily be synthesized in sufficientquantities to be used for tumor imaging by the new synthetic strategyshown in schemes 1 and 3

Scheme 2 outlines the synthesis of the anti-3-[¹⁸F]FACBC labelingprecursor, 1-tert-butylcarbamate-3-hydroxy-cyclobutane-1-carboxylicmethyl ester (9) and its conversion into anti-[¹⁸F]FACBC. Treatment of 9with trifluoromethane sulfonic anhydride/pyridine yielded the cis1-tert-butylcarbamate-3-trifluoromethanesulfonoxy-cyclobutane-1-carboxylicmethyl ester (10). Subsequent treatment of 10 with K F/K₂₂₂ followed byacid hydrolysis yielded anti-[¹⁸F]FACBC (12). In this syntheticstrategy, ˜20 mg of 10 must be used to obtain a reasonable conversion(˜24% radiochemical yield (RCY)) of 10 into anti-[¹⁸F]FACBC (12).

Scheme 3 outlines the synthesis of the anti-2-[¹⁸F]FACBC labelingprecursor,syn-4-(bis(4-methoxyphenyl)methyl)-2,3,4-oxathiazabicyclo[3.2.0]heptane-6-carboxylicacid ester 2-oxide (21) and its conversion into anti-2-[¹⁸F]FACBC.Treatment of 21 with K F/K₂₂₂ followed by acid hydrolysis will yieldanti-2-[¹⁸F]FACBC (22).

Scheme 4 outlines the synthesis of the anti-2-[¹⁸F]FACBC labelingprecursorsyn-4-(tert-butoxycarbonyl)-2,3,4-oxathiazabicyclo[3.2.0]heptane-6-carboxylicacid tert-butyl ester 2,2-dioxide (34) and its conversion intoanti-2-[¹⁸F]FACBC. Treatment of 34 with K¹⁸F/K₂₂₂ followed by acidhydrolysis will yield anti-2-[¹⁸F]FACBC (22).

Synthetic scheme 5 shows the synthesis of 2-substituted [¹⁸F]fluoroalkyl-1-aminocyclobutyl carboxylic acid, (S)-(−) anti-1-amino-2[¹⁸ F]fluoromethylcyclobutyl-1-carboxylic acid ((S)-(−)-anti-2-[¹⁸F]FMACBC)) and (R)-(+) anti-1-amino-2-[¹⁸F]fluoromethylcyclobutyl-1-carboxylic acid ((R)-(+)-anti-2-[¹⁸F]FMACBC)). The key feature of synthetic scheme 5 is the use of sulfonylester precursors such as 36 to enable high radiochemical incorporationof F-18 fluoride onto the 2-methyl group in high radiochemical yield andsubsequent efficient conversion of the radiolabeled intermediate [¹⁸F]36 into [¹⁸ F]37.

Schemes 6 through 9 depict synthetic routes for 2-substitutedradioiodinated derivatives of the present invention.

Synthetic scheme 6 delineates the preparation of (S)-(−)anti-1-amino-2-[¹²³I]iodocyclobutyl-1-carboxylic acid((S)-(−)-anti-2-[¹²³I]IACBC)) and (R)-(+)anti-1-amino-2-[¹²³I]iodocyclobutyl-1-carboxylic acid((R)-(+)-anti-2-[¹²³I]IACBC)). The key feature of synthetic scheme 6 isthe use of a cyclic sulfamidate precursor such as 21 to enableincorporation of I-123 iodide onto the 2-position i.e. (38) inpotentially>90% radiochemical yield.

Schemes 7 through 9 depict synthetic routes for 2-substitutedradioiodinated iodovinyl derivatives. Iodovinyl derivatives haveenhanced in vivo metabolic stability in comparison to iodoalkylderivatives i.e. 38 due to the attachment of the iodine to a sp2hydridized carbon. Synthetic scheme 7 shows the preparation of(S)-(−)anti-1-amino-2-[¹²³I]iodomethylenecyclobutyl-1-carboxylic acid((S)-(−)-anti-2-[¹²³I]IVACBC)) and(R)-(+)anti-1-amino-2-[¹²³I]iodomethylenecyclobutyl-1-carboxylic acid((R)-(+)-anti-2-[¹²³I]IVACBC)). The key feature of synthetic scheme 6 isthe use of only 100 μg an organotin precursor such as 42 to enableincorporation of I-123 iodide onto the 2-position in potentially >70%radiochemical yield.

Schemes 8 and 9 depict synthetic routes for 2-substituted radioiodinatediodoethenyl derivatives. Synthetic scheme 8 shows the preparation ofcis-(S)-(−) anti-1-amino-2-[¹²³I]iodoethenylcyclobutyl-1-carboxylic acid(cis-(S)-(−)-anti-2-[¹²³I]IEACBC)) and(cis-(R)-(+)anti-1-amino-2-[¹²³I]iodoethenylcyclobutyl-1-carboxylic acid((R)-(+)-anti-2-[¹²³I]IEACBC)). The key feature of synthetic scheme 7 isemploying a wittig reagent to obtain the cis confirmation of theiodovinyl moiety. Synthetic scheme 9 shows the preparation oftrans-(S)-(−)anti-1-amino-2-[¹²³I]iodoethenylcyclobutyl-1-carboxylicacid (trans-(S)-(−)-anti-2-[¹²³ I]IEACBC)) and(trans-(R)-(+)anti-1-amino-2-[¹²³I]iodoethenylcyclobutyl-1-carboxylicacid ((R)-(+)-trans-2-[¹²³I]IEACBC)). The key feature of syntheticscheme 9 is employing tri-n-butyltin hydride and AIBN reagents togenerate the trans conformation of the iodovinyl moiety.

Schemes 10 and 11 depict synthetic routes for 2-substituted Tc-99mderivatives of the present invention.

EXAMPLES

The amino acid compounds of the invention synthesized according to themethods disclosed herein are characterized by spectral analyses.Spectral analyses include fast atom bombardment mass spectrometry and ¹Hnuclear magnetic resonance spectrometry analysis. Followingpurification, all unlabeled agents are analyzed for chemical purity bythin-layer chromatography. All radiolabeled agents are analyzed forradiohomogeneity by thin layer radiochromatographic techniques that areknown in the art.

Example 1 Synthesis of 2-FACBC syn-5-(2-benzyloxycyclobutane)hydantoin(27) and anti-5-(2-benzyloxycyclobutane)hydantoin (28)

To a solution of 4 eq of ammonium carbonate (1.97 g, 20.5 mmoles) and 2eq of ammonium chloride (0.55 g, 10.2 mmoles) in 50 mL of water wasadded 1 eq of the cyclobutanone 26 (0.9 g, 5.11 mmoles) in 50 mL ofethanol. After stirring at room temperature for 15 minutes, a 1.2 eqportion of potassium cyanide (0.4 g, 6.1 mmoles) was added, and thereaction mix was heated at 70° C. overnight. The solvent was removedunder reduced pressure, and the crude cream colored solid was rinsedthoroughly with water to remove salts. The product (0.7 g, 51%) wasobtained as a 9:1 mixture of syn:anti isomers.

syn-1-(N-(tert-butoxycarbonyl)amino)-2-benzyloxycyclobutane-1-carboxylicacid (30)

A suspension of compounds 27 and 28 (1.26 g, 5.1 mmoles) in 12 mL of 3Nsodium hydroxide was heated at 110-115° C. overnight in a sealedstainless steel vessel. After cooling, the reaction mix was neutralizedto pH 6-7 with concentrated hydrochloric acid. After evaporation ofwater under reduced pressure, the resulting solid was extracted with hotethanol. The combined ethanol extracts were concentrated, and theresidue was dissolved in 50 mL of 9:1 methanol:triethylamine. To thesolution was added a 1.5 eq portion of di-tert-butyl dicarbonate (1.67g), and the solution was stirred at room temperature for 72 h. Thesolvent was removed under reduced pressure, and the crude product wasstirred in a mixture of ice-cold 80 mL of ethyl acetate and ice-cold 80mL of 0.2N hydrochloric acid for five minutes. The organic layer wasretained, and the aqueous phase was extracted with 2×80 mL of ice-coldethyl acetate. The combined organic layers were washed with 3×60 mL ofwater followed by usual work up. The N-Boc acid 30 (887 mg, 54%) wasobtained as a light yellow oil suitable for use in the next step withoutfurther purification.

syn-1-(N-(tert-butoxycarbonyl)amino)-2-benzyloxycyclobutane-1-carboxylicacid tert-butyl ester (31)

A 2.5 eq portion of tert-butyl 2,2,2-trichloroacetamide (1.5 g, 6.9mmol) was added to a solution of N-Boc acid 30 (887 mg, 2.76 mmoles) in10 mL of dichloromethane. After 2 days of stirring, the reaction mixturewas filtered, washed with dichloromethane and the filtrate concentratedunder reduced pressure, and the crude product was purified via silicagel column chromatography (1:8 ethyl acetate:hexane). The N-Boctert-butyl ester 31 (634 mg, 61%) was obtained as a colorless oil.

syn-1-(N-(tert-butoxycarbonyl)amino)-3-hydroxycyclobutane-1-carboxylicacid tert-butyl ester (32)

To a solution of 31 (350 mg, 0.93 mmoles) in 10 mL of CH₃OH under anargon atmosphere was added 105 mg of 10% Pd/C. The reaction mix wasstirred overnight at room temperature under a hydrogen atmosphere. Thesuspension was then filtered over Celite® and concentrated under reducedpressure purification via silica gel column chromatography (4:1 ethylacetate:hexane), which provided the alcohol 32 (269 mg, 100%) as a clearoil.

syn-4-(tert-butoxycarbonyl)-2,3,4-oxathiazabicyclo[3.2.0]heptane-6-carboxylicacid tert-butyl ester 2-oxide (33)

A solution of the N-Boc alcohol 32 (48 mg, 0.17 mmol) was added to acooled (−40° C.) solution of 2.5 eq of thionyl chloride (50 mg, 30 μL)in 1 mL acetonitrile under an argon atmosphere followed by the additionof 5 eq of pyridine (50 mg, 68 μL) in 0.5 mL of acetonitrile. After 10minutes the cooling bath was removed, and the reaction was continued for30 minutes. The reaction mix was partitioned between 10 mL of EtOAc and10 mL of H₂O. The aqueous layer was further extracted with 3×10 mL ofEtOAc. The organic layers were combined and washed with 20 mL of brinefollowed by usual work up. Silica gel column chromatography (12.5% EtOAcin hexane) afforded cyclic sulfamidite 33 as a colorless oil (45 mg,80%).

syn-4-(tert-butoxycarbonyl)-2,3,4-oxathiazabicyclo[3.2.0]heptane-6-carboxylicacid tert-butyl ester 2,2-dioxide (34)

A solution of the sulfamidite 33 (15 mg, 0.045 mmol) in 2 mL of CH₃CNwas cooled in an ice bath and treated successively with 1.1 eq of NaIO₄(11 mg), a catalytic amount of RuO₂.H₂O (˜0.1 mg) and 1.2 mL of H₂O.After 30 minutes of stirring, the ice bath was removed, and the reactionwas continued for 20 minutes. The reaction mixture was diluted in 10 mLof EtOAc and washed with 10 mL of saturated NaHCO₃ solution. The aqueouslayer was extracted with 2×10 mL of EtOAc, and the combined organiclayers were washed with 10 mL brine followed by usual work up. The crudeproduct was purified by silica gel column chromatography (25% EtOAc inhexane) to provide the cyclic sulfamidate 34 as a clear oil (15 mg,96%).

Preparation of (R,S)anti-1-amino-2-[¹⁸F]fluorocyclobutyl-1-carboxylicacid ((R,S) anti-2-[¹⁸F]FACBC), 22

To a glass vessel containing 610 mCi of no-carrier-added [¹⁸F]HF (30 μA,30 minute bombardment, theoretical specific activity of 1.7 Ci/nmole) in0.6 mL H₂O containing 5 mg of K₂CO₃ was added a 1 mL solution of 5 mgK₂₂₂ Kryptofix in CH₃CN. The solvent was removed at 110° C. with argongas flow, and an additional 1 mL of CH₃CN was added followed byevaporation with argon flow. This drying was repeated a total of 3 timesto remove residual H₂O. A 2-5 mg portion of the cyclic sulfamidateprecursor 34 in 1 mL of dry CH₃CN was added to the vial, and thereaction mix was heated at 90° C. for 10 minutes. The solvent wasremoved at 115° C. with argon gas flow, and the intermediate product wastreated with 0.5 mL of 4N HCl at 110° C. for 10 minutes. The aqueoushydrosylate was allowed to cool for 1 minute and then diluted withapproximately 4 mL of sterile saline. The aqueous solution was thentransferred to an ion retardation (IR) column assembly consisting of a7×120 mm bed of AG 11 A8 ion retard resin, a neutral alumina SepPak Plus(preconditioned with 10 mL water) and an HLB Oasis cartridge(preconditioned with 10 mL ethanol then blown dry with 20 mL air), andrinsed with 60 mL of sterile water and then attached to a dose vial. Theproduct [¹⁸F]22 was eluted in series through the ion retard resin, thealumina SepPak Plus and the HLB Oasis cartridge. The elution wasperformed with three successive portions of ˜4 mL sterile salinetransferred from the glass vial to the IR column assembly. Theradiolabeled product eluting from the column assembly passed through a0.22 μm sterile filter into a dose vial.

In all radiosyntheses, the only peak present on radiometric TLC analysiscorresponded to 22 and the radiochemical purity of the product exceeded99%. The isolated radiochemical yield (30%, non-optimized) wasdetermined using a dose-calibrator (Capintec CRC-712M).

Example 2 Tumor Binding Specificity of Inventive Compounds

The inventive compounds have been evaluated in vitro for tumor bindingspecificity (i.e. uptake cells) using a variety of tumor cell linesavailable in the art, along with reference compounds such as Me-AIB andBCH. Detailed description of these assays can be found in Martarello etal. (2002) Journal of Medicinal Chemistry, 45:2250-2259 and McConathy etal. (2003) Nuclear Medicine and Biology, 30:477-490. These, so called“amino acid uptake studies” are typically carried out with radiolabeledcompounds in at least five phenotypically different human tumor celllines (e.g. A549 lung carcinoma, MB468 breast carcinoma, DU145 prostatecarcinoma, SKOV3 ovarian carcinoma, and U87 glial blastoma). These tumorcell lines can be grown either in vitro or in vivo with severe combinedimmunodeficiency (SCID) mice as a host. The fore-mentioned tumor celllines are available at the Winship Cancer Institute of Emory University.

For the in vitro amino acid uptake studies, all cells can be grown tomonolayer confluency in T-175 culture flasks [Corning, Corning, N.Y.](approx. 1×10⁸ cells/flask) in Dulbecco's Modified Eagle's Medium (DMEM)[Sigma, St. Louis, Mo.] in a humidified incubator (370 C, 5% CO₂/95%air). Media are supplemented with 10% fetal calf serum [Hyclone, Logan,Utah], and antibiotics (10,000 U/ml penicillin and 10 mg/mlstreptomycin) (Sigma, St. Louis, Mo.). For tissue culture passage,monolayer cells are detached by gentle trypsinization, resuspended incomplete media, and split 1:10 into new T-flasks. Cultures are passagedweekly, and fed fresh media every 2 to 3 days. To initiate tumor growthin SCID mice, 1×10⁶ cells are injected s.c. bilaterally into the flanks(inguinal region) of the recipient animals using a 1 ml syringe with a27 gauge needle. Ex vivo experiments can be performed with animalscontaining tumors weighing between 500 mg and 1 g, as estimated bycaliper measurement (tumor weight=(π/6)*abc, where a, b and c are thetumor length, width and height, respectively).

In the in vitro studies the uptake rate of each amino acid compound ismeasured in each tumor line, as well as the dominant transportmechanisms of each tumor cell line. After trypsinization, cells areresuspended in serum-free media, then counted on a hemocytometer, withviability assessed through Trypan blue staining. Approximately 1×10⁷cells are exposed to each compound (15 μCi) in 15 ml of amino acid freemedia for 5, 10, 15, 30 and 60 minutes at 370 C. Cells are thencentrifuged at 150×g for 5 minutes, rinsed in 5 ml cold-saline,recentrifuged, resuspended in 3 ml saline, and placed into 12×75 mmglass vials (Fisher, Pittsburgh, Pa.). The vials are placed in aCobra-II gamma counter (Packard, Meriden, Conn.), with the activity percell number determined. Inhibition studies determine the dominanttransport mechanism (L, A or ASC) for each line [Martarello et al.(2002) supra; McConathy et al. (2003) supra]. For these studies, cellsare exposed to the compounds for 30 minutes in amino acid free mediacontaining one of three inhibitors (2-amino-norbornyl-2-carboxylic acid(BCH), 10 mM; α-(methylamino)-isobutyric acid (MeAIB), 10 mM; and analanine-serine-cysteine mixture 1:1:1, 10 mM). Saline washes areperformed as described above, and the filtered cells radioactivitydetermined on the gamma counter. Comparisons with the 30 minute controluptake indicate the major transporters used.

The compounds of the invention are further evaluated for their tumorspecificity and selectivity in tumor-bearing animal models. One canevaluate and compare the transport, accumulation and tissue distributionof each compound in these in vivo animal studies.

Tissue distribution of the compounds is measured in SCID mice (averageweight, 20-25 g) bearing human tumors as follows. The candidateradioligands (20 μCi in 0.4 ml 0.9% NaCl) are injected into the tailvein of tumor-bearing mice. The animals are sacrificed (cervicaldislocation) at 5, 30, 60 and 120 minutes post-injection. Tissues(blood, heart, liver, lungs, kidneys, bone, thyroid, muscle, brain andtumor) are excised, rinsed in saline, and blotted dry. The tissues areweighed, placed into 12×75 mm glass vials, the radioactivity determinedwith a gamma counter, and the percent dose/gram calculated. Totalactivities of blood and muscle are calculated by assuming that theyaccount for 7% and 40% of the total body mass, respectively. TABLE 1 %Dose Uptake/0.5e6 Cells of Anti-R,S 2-[¹⁸F]FACBC In Human Tumor CellsMDA DU145 SKOV3 U87 A549 MB468 No inhibitor 6.40 +/− 0.60 2.16 +/− 0.148.28 +/− 0.45 8.89 +/− 0.36 7.37 +/− 0.21 BCH 1.78 +/− 0.20 1.74 +/−0.01 2.48 +/− 0.23 3.99 +/− 0.39 1.67 +/− 0.36 MeAIB 2.07 +/− 0.26 0.75+/− 0.01 5.41 +/− 0.64 6.93 +/− 0.20 3.02 +/− 0.55

TABLE 2 % Dose/g DU145 Prostate Tumor Cells Implanted SC in SCID Miceblood heart lung liver pancreas spleen kidney muscle brain tumor boneMinutes Average 2.79 2.07 1.35 2.32 9.02 2.37 6.8 0.83 0.16 2.94 1.05 15(n = 5) Std. Dev. 0.81 1.66 3.38 0.72 24.70 4.99 2.81 0.33 0.05 1.200.56 Average 1.85 0.82 2.11 2.08 19.79 2.74 4.49 1.99 0.11 3.54 1.62 30(n = 5) Std. Dev. 0.07 0.20 1.39 0.24 5.24 1.23 0.27 0.15 0.06 0.80 0.56Average 0.80 1.32 0.18 1.31 2.57 0.84 1.85 0.25 0.17 2.56 1.22 60 (n =5) Std. Dev. 0.12 0.18 0.28 0.42 13.9 0.87 0.52 0.25 0.02 0.47 0.2Average 0.38 0.82 0.15 0.15 5.0 0.87 0.79 0.17 0.12 1.84 0.65 120 (n =5)  Std. Dev. 0.08 0.20 0.43 0.12 197 0.33 0.283 0.71 0.02 0.19 .09

TABLE 3 % Dose/g SKOV3 Ovarian Tumor Cells Implanted SC in SCID Miceblood heart lung liver pancreas spleen kidney muscle brain tumor boneMinutes Average 3.01 2.37 3.96 2.62 34.27 5.00 8.70 1.14 0.18 4.89 1.4015 (n = 5) Std. Dev. 0.81 0.80 1.13 1.25 17.65 2.16 3.09 0.49 0.06 2.390.67 Average 2.22 2.16 2.65 2.49 31.88 1.22 6.12 0.21 0.21 1.57 1.41 30(n = 5) Std. Dev. 0.27 1.29 0.29 0.33 7.96 5.26 2.82 0.19 0.07 5.06 0.66Average 0.14 0.40 1.31 1.28 17.57 2.67 0.27 1.03 0.14 1.58 0.91 60 (n =5) Std. Dev. 0.94 1.34 0.36 0.26 14.24 1.61 1.30 0.29 0.05 4.55 0.13Average 0.13 0.00 0.68 0.17 3.42 1.54 0.87 0.15 0.01 3.39 0.34 120 (n =5)  Std. Dev. 0.50 0.19 0.21 0.65 0.07 0.05 0.58 0.67 0.06 0.59 0.98

TABLE 4 % Dose/g A549 Lung Tumor Cells Implanted SC in SCID Mice bloodheart lung liver pancreas spleen kidney muscle brain tumor bone MinutesAverage 3.49 0.70 0.53 3.15 48.45 7.90 10.11  1.10 0.26 5.11 2.06 15 (n= 5) Std. Dev. 1.31 0.26 4.27 0.66 47.69 2.01 1.00 0.38 0.02 0.85 1.43Average 0.28 1.21 3.03 2.75 4.17 1.15 5n   1.43 0.15 4.28 0.11 30 (n =5) Std. Dev. 2.12 2.23 1.21 1.50 20.67 5.62 1.45 0.92 0.26 2.93 1.63Average 0.48 2.81 0.48 0.48 34.63 1.82 0.45 0.30 0.03 0.04 0.13 60 (n =5) Std. Dev. 0.72 0.46 0.36 0.46 2.03 2.08 2.14 0.18 0.12 3.59 0.07Average 0.00 0.35 0.56 0.58 3.20 1.42  1.233 0.07 0.01 0.31 0.62 120 (n= 5)  Std. Dev. 0.02 0.02 0.06 0.03 9.89 0.43 0.02 0.76 0.04 0.40 0.07

TABLE 5 % Dose/g MB468 Breast Tumor Cells Implanted SC in SCID Miceblood heart lung liver pancreas spleen kidney muscle brain tumor boneMinutes Average 3.26 2.21 .02 2.51 36.85 4.47 8.62 1.29 0.23 2.71 1.4615 (n = 5) Std. Dev. 0.34 0.35 4.42 0.51 9.50 1.15 0.96 0.35 0.05 1.740.40 Average 2.39 1.89 0.98 2.49 35.25 4.13 6.49 1.12 0.20 2.83 1.30 30(n = 5) Std. Dev. 10.35 0.18 1.23 1.26 1.1 0.95 1.09 0.25 .15 0.10 3Average 11 0.27 36 0.19 9.20 3.06 2.27 1.3 0.03 2.74 0.13 60 (n = 5)Std. Dev. 0.00 0.40 .07 .20 29.4 1.05 0.54 0.07 0.20 1.49 0.91 Average06 0.19 0.52 0.16 1.96 6 0.82 0.16 0 .36 0.14 120 (n = 5)  Std. Dev.0.35 .63 0.9 0.1 7.95 0.1 0.29 0.69 0 0 0.79

TABLE 6 % Dose/g U87 Glioma Tumor Cells Implanted SC in SCID Mice bloodheart lung liver pancreas spleen kidney muscle brain tumor bone MinutesAverage .20 2.29 4.01 4 49.8 61.63 1.21 14 .2e .4 2.10 15 (n = 5) Std.Dev. 3.11 2.01 01 0 .76 1.08 9.0 0.12 .01 .9 0.4 Average 2.10 0.50 22.23 4.0 1 2.55 2.08 1.12 0.8 0 30 (n = 5) Std. Dev. 0.27 1.16 1.29 0.7117.03 0.09 5.0 0.04 .1 0.41 1.66 Average 0.51 0.13 0.18 1.58 2.38 3.42 910.20 016 0.60 1.40 60 (n = 5) Std. Dev. 0.06 0.12 0.04 0.20 1.27 3.400.45 0.94 0.07 .13 0.22 Average 0.48 0.30 0.20 0.78 0.20 45 0.10 0.390.3 2.7 12 120 (n = 5)  Std. Dev. 0.82 0.89 0.8 0.15 5.2 0.48 0.37 0.90.00 0.65 1.04

The inventive amino acid compounds have several advantages over[¹¹C]-methionine as well as [¹⁸F]-fluorodeoxyglucose (FDG) for clinicalimaging of tumors. These advantages are related to: 1)(S)-(−)-anti-2-[¹⁸F]FACBC and (R)-(+)-anti-2-[¹⁸F]FACBC are notmetabolized by endogenous enzymes, and radiolabeled metabolites will notconfound the interpretation of the images as can be the case with[¹¹C]methionine; 2) (S)-(−)-anti-2-[¹⁸F]FACBC and(R)-(+)-anti-2-[¹⁸F]FACBC are likely to have better transport,accumulation and tumor imaging characteristics compared to[¹¹C]methionine; 3) (S)-(−)-anti-2-[¹⁸F]FACBC and(R)-(+)-anti-2-[¹⁸F]FACBC imaging of tumors, will provide different andmore clinically useful information than that obtained with[¹⁸F]-fluoro-2-deoxy-D-glucose (FDG); 4) labeling alicyclic/branchedcandidate amino acids with [¹⁸F] instead of [¹¹C] will providesubstantial logistical and cost-effective benefits for clinical imagingof tumors in a busy nuclear medicine department, due to the longerhalf-life of [¹⁸F] (t_(1/2)=110 min) compared to [¹¹C] (t_(1/2)=20 min):and 5) (S)-(−)-anti-2-[¹⁸F]FACBC and (R)-(+)-anti-2-[¹⁸F]FACBC haveadvantages over anti-3-[¹⁸F]FACBC and syn-3-[¹⁸F]FACBC because asignificantly higher radiochemical yield is possible due to the morereactive and less moisture sensitive cyclic sulfamidate radiolabelingprecursors in comparison to more moisture sensitive trifluoromethanesulfonic ester radiolabeling precursors used in the synthesis ofanti-3-[¹⁸F]FACBC.

As exemplified above, the amino acid compounds of the invention haveadvantageous physiological characteristics (i.e., tumor bindingspecificity and selectivity, in vivo stability etc). Anti-2-[¹⁸F]FACBCis an excellent tumor imaging agent using PET based on the followingdata: 1) In vitro studies in human U87 glioma cells, human DU145prostate cancer cells, A549 lung cancer cells, SKOV3 ovarian cancercells, and MB468 breast cancer cells (Table 1) demonstrate thatanti-2-[¹⁸F]FACBC shows high uptake by the type “L” type large-neutralamino acid transport system; 2) In vivo biodistribution studies (Tables2-6) performed in A549 (lung), DU145 (prostate), SKOV3 (ovary), and MDAMB468 (breast) tumor-bearing SCID mice with anti-2-[¹⁸F]FACBC injectedintravenously showed a rapid and prolonged accumulation of radioactivityin tumors with good tumor uptake (1.4-3.4% dose/g), tumor-to muscleratio (2-4), tumor-to brain ratio (15-30), tumor-to kidney ratio(1.5-2.7) and tumor-to liver ratio (2.4-4) at 120 min p.i. These dataindicate that anti-2-[¹⁸F]FACBC can provide clinically useful data inboth brain and systemic tumors.

The present invention also includes stereoisomers as well as opticalisomers, e.g. mixtures of enantiomers as well as individual enantiomersand diastereomers which arise as a consequence of structural asymmetry.

The compounds of formulas I and II may also be solvated, especiallyhydrated. Hydration may occur during manufacturing of the compounds orcompositions comprising the compounds, or the hydration may occur overtime due to the hygroscopic nature of the compounds. In addition, thecompounds of the present invention can exist in unsolvated as well assolvated forms with pharmaceutically acceptable solvents such as water,ethanol, and the like. In general, the solvated forms are consideredequivalent to the unsolvated forms for the purposes of the presentinvention.

When the compounds of the invention are to be used as imaging agents,they must be labeled with suitable radioactive halogen isotopes such as¹²³I, ¹³¹I, ¹⁸F, ⁷⁶Br, and ⁷⁷Br. The radiohalogenated compounds of thisinvention can easily be provided in kits with materials necessary forimaging a tumor. For example, a kit can contain a final product labeledwith an appropriate isotope (e.g. ¹⁸F) ready to use for imaging or anintermediate compound (e.g. compound 1a or 1b in scheme 1) and a label(e.g. K[¹⁸F]F) with reagents (e.g. solvent, deprotecting agent) suchthat a final product can be made at the site or time of use.

In the first step of the present method of imaging, a labeled compoundof formula I or II is introduced into a tissue or a patient in adetectable quantity. The compound is typically part of a pharmaceuticalcomposition and is administered to the tissue or the patient by methodswell known to those skilled in the art. For example, the compound can beadministered either orally, rectally, parenterally (intravenous, byintramuscularly or subcutaneously), intracistemally, intravaginally,intraperitoneally, intravesically, locally (powders, ointments ordrops), or as a buccal or nasal spray.

In an imaging method of the invention, the labeled compound isintroduced into a patient in a detectable quantity and after sufficienttime has passed for the compound to become associated with tumor tissuesor cells, the labeled compound is detected noninvasively inside thepatient. In another embodiment of the invention, a labeled compound offormula I or II is introduced into a patient, sufficient time is allowedfor the compound to become associated with tumor tissues, and then asample of tissue from the patient is removed and the labeled compound inthe tissue is detected apart from the patient. Alternatively, a tissuesample is removed from a patient and a labeled compound of formula I orII is introduced into the tissue sample. After a sufficient amount oftime for the compound to become bound to tumor tissues, the compound isdetected. The term “tissue” means a part of a patient's body. Examplesof tissues include the brain, heart, liver, blood vessels, and arteries.A detectable quantity is a quantity of labeled compound necessary to bedetected by the detection method chosen. The amount of a labeledcompound to be introduced into a patient in order to provide fordetection can readily be determined by those skilled in the art. Forexample, increasing amounts of the labeled compound can be given to apatient until the compound is detected by the detection method ofchoice. A label is introduced into the compounds to provide fordetection of the compounds.

The administration of the labeled compound to a patient can be by ageneral or local administration route. For example, the labeled compoundmay be administered to the patient such that it is delivered throughoutthe body. Alternatively, the labeled compound can be administered to aspecific organ or tissue of interest.

Those skilled in the art are familiar with determining the amount oftime sufficient for a compound to become associated with a tumor. Theamount of time necessary can easily be determined by introducing adetectable amount of a labeled compound of formula I or II into apatient and then detecting the labeled compound at various times afteradministration.

Those skilled in the art are familiar with the various ways to detectlabeled compounds. For example, magnetic resonance imaging (MRI),positron emission tomography (PET), or single photon emission computedtomography (SPECT) can be used to detect radiolabeled compounds. PET andSPECT are preferred when the compounds of the invention are used astumor imaging agents. The label that is introduced into the compoundwill depend on the detection method desired. For example, if PET isselected as a detection method, the compound must possess apositron-emitting atom, such as ¹¹C or ¹⁸F.

The radioactive diagnostic agent should have sufficient radioactivityand radioactivity concentration which can assure reliable diagnosis. Forinstance, in case of the radioactive metal being technetium-99m, it maybe included usually in an amount of 0.1 to 50 mCi in about 0.5 to 5.0 mlat the time of administration. The amount of a compound of formula maybe such as sufficient to form a stable chelate compound with theradioactive metal.

The inventive compound as a radioactive diagnostic agent is sufficientlystable, and therefore it may be immediately administered as such orstored until its use. When desired, the radioactive diagnostic agent maycontain any additive such as pH controlling agents (e.g., acids, bases,buffers), stabilizers (e.g., ascorbic acid) or isotonizing agents (e.g.,sodium chloride). The imaging of a tumor can also be carried outquantitatively using the compounds herein so that a therapeutic agentfor a given tumor can be evaluated for its efficacy.

Preferred compounds for imaging include a radioisotope such as ¹²³I,¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁸F, ⁷⁶Br, ⁷⁷Br or ¹¹C.

The synthetic schemes described herein represent exemplary syntheses ofpreferred embodiments of the present invention. However, one of ordinaryskill in the art will appreciate that starting materials, reagents,solvents, temperature, solid substrates, synthetic methods, purificationmethods, analytical methods, and other reaction conditions other thanthose specifically exemplified can be employed in the practice of theinvention without resort to undue experimentation. All art-knownfunctional equivalents, of any such materials and methods are intendedto be included in this invention. The terms and expressions which havebeen employed are used as terms of description and not of limitation,and there is no intention that in the use of such terms and expressionsof excluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers and enantiomers of the group members, are intended to beindividually included. When a Markush group or other grouping is usedherein, all individual members of the group and all combinations andsubcombinations possible of the group are intended to be individuallyincluded in the disclosure. When a compound is described herein suchthat a particular isomer or enantiomer of the compound is not specified,for example, in a formula or in a chemical name, that description isintended to include each isomers and enantiomer of the compounddescribed individually or in any combination. Additionally, unlessotherwise specified, all isotopic variants of compounds disclosed hereinare intended to be encompassed by the disclosure. For example, it willbe understood that any one or more hydrogens in a molecule disclosed canbe replaced with deuterium or tritium. Isotopic variants of a moleculeare generally useful as standards in assays for the molecule and inchemical and biological research related to the molecule or its use.Specific names of compounds are intended to be exemplary, as it is knownthat one of ordinary skill in the art can name the same compoundsdifferently.

Many of the molecules disclosed herein contain one or more ionizablegroups [groups from which a proton can be removed (e.g., —COOH) or added(e.g., amines) or which can be quaternized (e.g., amines)]. All possibleionic forms of such molecules and salts thereof are intended to beincluded individually in the disclosure herein. With regard to salts ofthe compounds herein, one of ordinary skill in the art can select fromamong a wide variety of available counterions, those that areappropriate for preparation of salts of this invention for a givenapplication.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their filing date and it is intended that this information can beemployed herein, if needed, to exclude specific embodiments that are inthe prior art. For example, when a compound is claimed, it should beunderstood that compounds known and available in the art prior toApplicant's invention, including compounds for which an enablingdisclosure is provided in the references cited herein, are not intendedto be included in the composition of matter claims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

All references cited herein are hereby incorporated by reference to theextent that there is no inconsistency with the disclosure of thisspecification. In particular, U.S. Pat. Nos. 5,817,776, 5,808,146, andWO 03/093412 are cited herein and incorporated by reference herein toprovide examples of the amino acid analogs that can be made using theinvention and the detailed synthetic methods. Some references providedherein are incorporated by reference to provide details concerningsources of starting materials, additional starting materials, additionalreagents, additional methods of synthesis, additional methods ofanalysis and additional uses of the invention.

1. An amino acid analog having the general formula:

Wherein Y is selected from the group consisting of (CR₅,R₆)n, n=1-4, N,O, S, and Se; X is selected from the group consisting of halogen,haloalkyl, halocycloalkyl, halocycloalkenyl, halocycloalkynyl, haloacyl,haloaryl, haloheteroaryl, haloalkenyl, haloalkynyl, Tc-99m chelate andRe chelate, where halo or halogen in X is selected from the groupconsisting of F, Cl, Br, I, At, F-18, Br-76, 1-123, 1-124; R4, R₅ and R₆are independently selected from the group consisting of H, alkyl,haloalkyl, cycloalkyl, halocycloalkyl, heteroaryl, aryl, haloaryl,haloheteroaryl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, where halois non-radioactive F, Cl, Br and I; R₁ and R₂ are independently selectedfrom the group consisting of H, alkyl, haloalkyl, cycloalkyl,halocycloalkyl, cycloalkenyl, halocycloalkenyl, cycloalkynyl,halocycloalkynyl, acyl, haloacyl, aryl, haloaryl, heteroaryl,haloheteroaryl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, Tc-99m andRe chelates; R₃ is selected from the group consisting of H, alkyl,haloalkyl, cycloalkyl, halocycloalkyl, cycloalkenyl, halocycloalkenyl,cycloalkynyl, halocycloalkynyl, acyl, haloacyl, aryl, haloaryl,heteroaryl, haloheteroaryl, alkenyl, haloalkenyl, alkynyl, andhaloalkynyl; or a pharmaceutically acceptable salt thereof.
 2. The aminoacid analog of claim 1 wherein X is selected from the group consistingof halo, haloalkyl_(C1-C6), halocycloalkyl, halocycloalkenyl,halocycloalkynyl, haloacyl, haloaryl, haloheteroaryl,haloalkenyl_(C1-C6), and haloalkynyl_(C1-C6).
 3. The amino acid analogof claim 2 wherein R₁, R₂, R₃, R₄, R₅, and R₆ are selected independentof each other from the group consisting of hydrogen, alkyl_(C1-C6),alkenyl_(C1-C6)and alkynyl_(C1-C6).
 4. The amino acid analog of claims 2or 3 wherein X is selected from the group consisting of halogen,haloalkyl_(C1-C6), haloalkenyl_(C1-C6), and haloalkynyl_(C1-C6), whereinhalo is either ¹⁸F or ¹²³I.
 5. The amino acid analog of claim 4 whereinX is halogen or haloalkyl_(C1-C4) and R₁, R₂, R₃, R₄, R₅, and R₆ areindependent of each other hydrogen or alkyl_(C1-C4).
 6. The amino acidanalog of claims 4 or 5 wherein Y is CH₂.
 7. The amino acid analog ofclaim 6 wherein the analog is 2-[¹⁸F]FACBC.
 8. The amino acid analog ofclaim 6 wherein the analog is 2-[¹²³I]IACBC.
 9. The amino acid analog ofclaim 6 wherein the analog is 2-[¹⁸F]FMACBC.
 10. The amino acid analogof claim 6 wherein the analog is 2-[¹²³I]IVACBC.
 11. The amino acidanalog of claim 6 wherein the analog is 2-[¹²³I]IEACBC.
 12. The aminoacid analog of claim 1 wherein R₁-R₆ are hydrogen, Y is CH₂, and X isTc99m.
 13. A compound of the formula:

wherein Y is selected from the group consisting of (CR₅,R₆)n, n=1-4, N,O, S, and Se; X is selected from the group consisting of halo,haloalkyl, halocycloalkyl, halocycloalkenyl, halocycloalkynyl, haloacyl,haloaryl, haloheteroaryl, haloalkenyl, haloalkynyl, Tc-99m and Rechelates, where halo in X is selected from the group consisting of F,Cl, Br, I, At, F-18, Br-76, I-123, and I-124; R₄, R₅ and R₆ areindependently selected from the group consisting of H, alkyl, haloalkyl,cycloalkyl, halocycloalkyl, heteroaryl, aryl, haloaryl, haloheteroaryl,alkenyl, haloalkenyl, alkynyl, and haloalkynyl, where halo isnon-radioactive F, Cl, Br and I; R₁ and R₂ are independently selectedfrom the group consisting of H, alkyl, haloalkyl, cycloalkyl,halocycloalkyl, cycloalkenyl, halocycloalkenyl, cycloalkynyl,halocycloalkynyl, acyl, haloacyl, aryl, haloaryl, heteroaryl,haloheteroaryl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, Tc-99m andRe chelates; R₃ is selected from the group consisting of H, alkyl,haloalkyl, cycloalkyl, halocycloalkyl, cycloalkenyl, halocycloalkenyl,cycloalkynyl, halocycloalkynyl, acyl, haloacyl, aryl, haloaryl,heteroaryl, haloheteroaryl, alkenyl, haloalkenyl, alkynyl, andhaloalkynyl; or a pharmaceutically acceptable salt thereof.
 14. Thecompound of claim 13 wherein X is selected from the group consisting ofhalogen, haloalkyl_(C1-C6), haloalkenyl_(C1-C6), andhaloalkynyl_(C1-C6).
 15. The compound of claim 14 wherein R₁, R₂, R₃,R₄, R₅, and R₆ are selected independent of each other from the groupconsisting of hydrogen, alkyl_(C1-C6), alkenyl_(C1-C6) andalkynyl_(C1-C6).
 16. The compound claim 15 wherein X is halogen orhaloalkyl_(C1-C4) and R₁, R₂, R₃, R₄, R₅, and R₆ are independent of eachother hydrogen or alkyl_(C1-C4).
 17. The compound of claim 16 wherein Xis halogen and R₁, R₂, R₃, R₄, R₅, and R₆ are hydrogen, and Y is CH. 18.The compound of claim 17 wherein X is ¹⁸F.
 19. The compound of claim 17wherein X is ¹²³I.
 20. A method of synthesizing an amino acid analogaccording to claim 1 wherein the method comprises the step of reacting acompound of formula III with reagents to yield the amino acid analog offormula I, wherein formula III is

wherein Y is selected from the group consisting of (CR₅,R₆)n, n=1-4, N,O S, and Se; R₄, R₅ and R₆ are independently selected from the groupconsisting of H, alkyl haloalkyl, cycloalkyl, halocycloalkyl,heteroaryl, aryl, haloaryl, haloheteroaryl, alkenyl, haloalkenyl,alkynyl, and haloalkynyl, where halo is selected from the groupconsisting of non-radioactive F, Cl, Br and I; R₁ is selected from thegroup consisting of H, alkyl haloalkyl, cycloalkyl, halocycloalkyl,cycloalkenyl, halocycloalkenyl, cycloalkynyl, halocycloalkynyl, acyl,haloacyl, aryl, haloaryl, heteroaryl, haloheteroaryl, alkenyl,haloalkenyl, alkynyl, haloalkynyl, Tc-99m and Re chelates; R₃ isselected from the group consisting of H, alkyl haloalkyl, cycloalkyl,halocycloalkyl, cycloalkenyl, halocycloalkenyl, cycloalkynyl,halocycloalkynyl, acyl, haloacyl, aryl, haloaryl, heteroaryl,haloheteroaryl, alkenyl, haloalkenyl, alkynyl, and haloalkynyl.
 21. Themethod of claim 20 wherein the amino acid analog is 2-[¹⁸F]FACBC.
 22. Adiagnostic composition for imaging a tumor, comprising a radiolabeledcompound of claim 1, and a pharmaceutically acceptable carrier.
 23. Thediagnostic composition of claim 22 wherein the labeled compound is2-[¹⁸F]FACBC.
 24. A method of tumor imaging by positron emissiontomography or single photon emission computed tomography, comprising: a)administering to a subject suspected of having a tumor animage-generating amount of a labeled compound of claim 1; b) allowingsufficient time for the labeled compound to become associated with thetumor; and c) measuring the distribution of the labeled compound in thesubject by PET or SPECT.
 25. The method of claim 24 wherein the labeledcompound is 2-[¹⁸F]FACBC.